A virtually complete absence of glutathione reductase activity was found in the erythrocytes of all three children (one male, two females) from a consanguineous marriage. Intermediate values were found in the erythrocytes of both parents. The enzyme activity could not be restored either by addition of FAD in vitro or by administration of riboflavin in vivo. The amount of reduced glutathione in the erythrocytes was normal in each case. Severely diminished glutathione stability during incubation with acetylphenylhydrazine was observed in the erythrocytes of the siblings, as well as intermediate stability in the parents' red cells. Clinically, this deficiency was manifested by hemolytic crises after eating fava beans in the eldest daughter (patient), and possibly by cataracts in her own and in her brother's eyes. Very low activities of glutathione reductase were also found in the leukocytes of this family: 13%-15% of normal values for the children and 64%-66% for the parents. Moreover, the same deficiency was found in the purified white blood cells of the propositus: 8% of normal values in the polymorphonuclear (PMN) cells, 4% in the lymphocytes, and 15% in the monocytes, together with 11% in the platelets. Finally, we found an abnormal oxygen consumption of the propositus' PMNs after phagocytosis of zymosan particles, suggesting that the glutathione reductase reaction was involved in the bactericidal capacity of these cells.
During a study on the separation of human blood monocytes from lymphocytes, a method was developed to recognize and count monocytes by electronic means. Lightscattering (Cytograf, Bio/Physics), and changes in electrical resistance (Channelyzer, Coulter) were used to size mononuclear leukocytes directly in cell suspensions. Both methods revealed a size distribution profile in which two populations of mononuclear leukocytes could be distinguished. The largest cells were virtually eliminated after phagocytosis of iron particles. We confirmed that these cells were monocytes by three different criteria: the intracellular lysozyme activity, the number of phagocytes, and the percentage of cells with kidney-shaped nuclei. The highly significant correlations we found showed that monocytes could be recognized and counted by electronic sizing. For this method, purified mononuclear leukocyte preparations had to be used, since the presence of erythrocytes, platelets, and polymorphonuclear cells interfered. Statistical analysis revealed that electronic sizing permitted discrimination of differences in monocyte content of 4.5%, with a probability of 95%. It was calculated that this sensitivity of electronic monocyte counting was about three times higher than the sensitivity of microscopic methods. Since 100,000 cells can be sized within a few seconds, not only the efficiency of the preparation but also minor changes in the size of monocytes and lymphocytes introduced during the isolation can be followed.
By separation of human blood lymphocyte suspensions on isotonic linear density gradients, fractions were obtained which differed in their response in vitro towards mitogens, antigens and allogeneic lymphocytes, as tested by incorporation of radioactive thymidine. Next, light ( p < 1.067 g/cm3) and heavy ( p > 1.067 g/cm3) lymphocytes were separated on discontinuous density gradients and their reactivity tested t o phytohemagglutinin, pokeweed mitogen, concanavalin A, purified protein derivative of tuberculin, a cocktail of antigens, and in the mixed lymphocyte culture (MLC). The light lymphocytes responded 1.6 t o 6 times more strongly than the heavy lymphocytes ( p < 0.0005) to all stimuli tested. The spontaneous thymidine incorporation of the light lymphocytes was 16 times as high as that of the heavy lymphocytes. In controls, the recombined light and heavy lymphocytes showed the same reactivity as the unseparated cell suspension. Furthermore, the stimulatory capacity in the MLC was significantly higher in the light lymphocyte fraction than in the heavy lymphocytes. Finally, the capacity t o recruit and t o be recruited in the lymphocyte reaction on antigens was also highest in the light lymphocytes.The light and heavy lymphocytes showed a similar dose response curve as well as time courses in the response t o all stimuli tested. Addition of purified monocytes to the heavy lymphocytes t o the same level as was found in the light lymphocyte fraction did not restore the level of reactivity to that of the light lymphocytes. The heavy and light lymphocytes contained identical proportions of T (E rosettes) B (IgG-bearing cells) and C3 receptor-bearing lymphocytes (EAC rosettes). The two fractions had similar cytotoxic capacity in antibody-dependent cytotoxicity.It is concluded that the reactivity differences are an intrinsic property of the heavy and light lymphocyte subsets.
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